anti-CD18 Monoclonal Antibody Research Articles

Preclinical development of SGN-CD47M: Protease-activated antibody technology enables selective tumor targeting of the innate immune checkpoint receptor CD47.

CD47 is a cell surface glycoprotein that is expressed on normal human tissues and has a key role as a marker of self. Tumor cells have coopted CD47 overexpression to evade immune surveillance and thus blockade of CD47 is a highly active area of clinical exploration in oncology. However, clinical development of CD47-targeted agents has been complicated by its robust expression in normal tissues and the toxicities that arise from blocking this inhibitory signal. Further, pro-phagocytic signals are not uniformly expressed in tumors and antibody blockade alone is often not sufficient to drive antitumor activity. The inclusion of an IgG1 antibody backbone into therapeutic design has been shown to serve as an additional pro-phagocytic signal but also exacerbates toxicities in normal tissues. Therefore, a need persists for more selective therapeutic modalities targeting CD47. To address these challenges, we developed SGN-CD47M, a humanized anti-CD47 IgG1 monoclonal antibody linked to novel masking peptides through linkers designed to be cleaved by active proteases enriched in the tumor microenvironment. Masking technology has the potential to increase the amount of drug that reaches the tumor microenvironment, while concomitantly reducing systemic toxicities. We demonstrate that SGN-CD47M is well tolerated in cynomolgus monkeys and displays a 20-fold improvement in tolerability to hematologic toxicities when compared to the unmasked antibody. SGN-CD47M also displays preferential activation in the tumor microenvironment that leads to robust single-agent antitumor activity. For these reasons, SGN-CD47M may have enhanced antitumor activity and improved tolerability relative to existing therapies that target the CD47-SIRPα interaction.

Cluster of differentiation-44 as a novel biomarker of lupus nephritis and its role in kidney inflammation and fibrosis.

CD44 is a transmembrane glycoprotein implicated in tissue inflammation and fibrosis. We investigated its role in kidney inflammation and fibrosis in a murine model of lupus nephritis (LN), and the clinico-pathological association of serum CD44 level in patients with biopsy-proven Class III/IV ± V LN. NZB/W F1 mice were treated with control IgG or anti-CD44 monoclonal antibody for 4 weeks and disease parameters assessed. Serum CD44 level in LN patients was determined by ELISA. Control groups included healthy subjects and patients with non-renal SLE or non-lupus renal disease. CD44 expression was absent in the normal kidney, but it was expressed in proximal and distal tubular epithelial cells and infiltrating cells in renal biopsies from patients with active proliferative LN. ScRNA-Seq datasets confirmed that CD44 was predominantly expressed in tubular cells and all immune cells identified in LN patients including tissue resident, inflammatory and phagocytic macrophages, Treg cells, effector and central memory CD4+ T cells, resident memory CD8+ T cells and naïve and activated B cells. Treatment of NZB/W F1 mice with anti-CD44 antibody preserved kidney histology and reduced proteinuria, tubulo-interstitial infiltration of CD3+, CD4+ and CD19+ immune cells, and mediators of kidney fibrosis compared to Control mice. Longitudinal studies showed that serum CD44 level increased prior to clinical renal flare by 4.5 months and the level decreased after treatment. ROC curve analysis showed that CD44 level distinguished patients with active LN from healthy subjects and patients with quiescent LN, active non-renal lupus, and non-lupus CKD (ROC AUC of 0.99, 0.96, 0.99 and 0.99 respectively). CD44 level correlated with leukocyte infiltration and interstitial inflammation scores in active LN kidney biopsies. Our findings suggest that CD44 plays a pathogenic role in renal parenchymal inflammation and fibrosis in active LN and monitoring CD44 may facilitate early diagnosis of flare.

A novel anti-CD47 antibody with therapeutic potential for NK/T-cell lymphoma

ABSTRACT NK/T-cell lymphoma (NKTCL) is a rare type of non-Hodgkin lymphoma (NHL). Although L-asparaginase-based chemotherapy has significantly improved survival in early-stage patients, the prognosis is poor in advanced and relapsed or refractory patients. CD47 is a promising target for cancer immunotherapy. However, the expression of CD47 in NKTCL and the antitumor effect and mechanism of the anti-CD47 monoclonal antibody (mAb) AK117 in NKTCL remain unclear. Firstly, the expression level of CD47 protein in NKTCL cells was detected by immunoblot and flow cytometry. Secondly, in order to validate the role of CD47 downregulation in the proliferation, apoptosis, and cell cycle of NKTCL cells, we used shRNA transfection to knock down CD47 expression. We determined the effect of knocking down CD47 and the novel anti‐CD47 antibody AK117 on the phagocytosis of NKYS and YTS cells by M2 macrophages in vitro. Finally, we assessed the ability of AK117 to inhibit tumor growth in an NKTCL xenograft model in which YTS cells were engrafted in SCID mice. The results showed that CD47 is relatively highly expressed in NKTCL cells. CD47 knockdown in NKTCL promoted phagocytosis by M2 macrophages in an in vitro coculture assay. The study also demonstrated that anti-CD47 mAb AK117 promoted phagocytosis of NKTCL cells by M2 macrophages. In addition, in vivo experiments showed that the anti-CD47 mAb AK117 significantly inhibited the growth of subcutaneous xenograft tumors in SCID mice compared to the control antibody IgG. Our results indicate that targeting CD47 monoclonal antibodies is a potential therapeutic strategy for NKTCL.

Evaluation of Tumor-Associated Macrophages and Micro-Vessel Density in Verrucous Carcinoma and Squamous Cell Carcinoma of the Oral Cavity.

Tumor-associated macrophages (TAMs) are a crucial cellular component of human tumors, but their exact roles in tumor growth and angiogenesis during cancer progression are still subject to debate. The present study aimed to explore the impact of TAMs on tumor progression and angiogenesis in oral squamous cell carcinoma (OSCC) and oral verrucous carcinoma (OVC). For this cross-sectional study, 60 samples consisting of 40 OSCC and 20 OVC samples were chosen. Immunohistochemical staining was performed on the samples using anti-CD68 and anti-CD31 monoclonal antibodies to evaluate the frequency of TAMs and micro-vessel density (MVD), respectively. The data were analyzed using statistical software SPSS (v. 23) and through the independent samples Student t-test, one-way analysis of variance, and Pearson's correlation coefficient. The results of our study revealed that the mean number of TAMs and MVD were significantly greater in OSCC samples compared to OVC samples (P = 0.001) (P = 0.004). Also, the average number of TAMs and MVD increased in the high grade of OSCC; however, the correlation between both parameters and histopathological grades of OSCC was significant only for MVD. Our study found no significant correlation between TAMs and MVD (P = 0.005). Based on the results of our study, both TAMs and MVD were found to be significantly higher in OSCC samples compared to OVC samples. These findings suggest that the progression of oral cancer is associated with an increase in TAMs and vascular density. Additionally, the use of CD68 and CD31 markers may help in differentiating between these two types of tumors.

Preclinical ImmunoPET Imaging Using a Zr-89-Labeled Anti-CD146 Monoclonal Antibody for Diagnosis of Melanoma.

The aim of this study was to evaluate the preclinical efficacy of [89Zr]Zr-DFO-Ab253 as a novel positron emission tomography (PET) tracer for CD146-positive malignant melanoma imaging. Considering the high expression of CD146 in malignant melanoma, this study investigated the effect of different CD146 expression levels on the tumor uptake of [89Zr]Zr-DFO-Ab253. CD146 selectivity was investigated by using the CD146-positive human melanoma cell A375 and the CD146-negative human alveolar epithelial cell A549. The cell uptake of [89Zr]Zr-DFO-Ab253 tracers was investigated, and receptor-binding affinities were measured by radioactive enzyme-linked immunosorbent assay. Biodistribution studies and micro-PET imaging of the radiotracers were performed on mice bearing A375 and A549 xenografts under baseline and blocking conditions. An immunohistochemical test was performed using A375 and A549 tissue sections for CD146 expression level analysis. [89Zr]Zr-DFO-Ab253 was obtained with a high radiochemical yield (87.86 ± 4.66%) and a satisfactory radiochemical purity (>98.0%). The specificity and affinity of [89Zr]Zr-DFO-Ab253 were confirmed in melanoma A375 cells and in vivo PET imaging of A375 tumor models. [89Zr]Zr-DFO-IgG and A549 lung tumors were prepared as control radiotracers and negative models to verify the specificity of [89Zr]Zr-DFO-Ab253 on CD146. [89Zr]Zr-DFO-Ab253 has a Kd of 4.01 ± 0.50 nM. PET imaging and biodistribution showed a higher uptake of [89Zr]Zr-DFO-Ab253 in A375 melanomas than that in A549 tumors (42.1 ± 4.04% vs 7.87 ± 1.30% ID/g at 120 h, P < 0.05). A low tumor uptake of [89Zr]Zr-DFO-IgG was observed with uptakes of 1.91 ± 0.41 and 2.80 ± 0.14 ID%/g when blocked at 120 h. The radiation-absorbed dose was calculated to be 0.13 mSv/MBq. This study demonstrates the synthesis and preclinical evaluation of [89Zr]Zr-DFO-Ab253 and indicates that the novel tracer has promising applications in malignant melanoma-specific PET imaging because of its high uptake and long-time retention in malignant melanoma. It also provides feasibility for the development of integrated molecular probes for diagnosis and treatment based on the CD146 target.

Phagocytosis Checkpoints in Glioblastoma: CD47 and Beyond.

Glioblastoma multiforme (GBM) is one of the deadliest human cancers with very limited treatment options available. The malignant behavior of GBM is manifested in a tumor which is highly invasive, resistant to standard cytotoxic chemotherapy, and strongly immunosuppressive. Immune checkpoint inhibitors have recently been introduced in the clinic and have yielded promising results in certain cancers. GBM, however, is largely refractory to these treatments. The immune checkpoint CD47 has recently gained attention as a potential target for intervention as it conveys a "don't eat me" signal to tumor-associated macrophages (TAMs) via the inhibitory SIRP alpha protein. In preclinical models, the administration of anti-CD47 monoclonal antibodies has shown impressive results with GBM and other tumor models. Several well-characterized oncogenic pathways have recently been shown to regulate CD47 expression in GBM cells and glioma stem cells (GSCs) including Epidermal Growth Factor Receptor (EGFR) beta catenin. Other macrophage pathways involved in regulating phagocytosis including TREM2 and glycan binding proteins are discussed as well. Finally, chimeric antigen receptor macrophages (CAR-Ms) could be leveraged for greatly enhancing the phagocytosis of GBM and repolarization of the microenvironment in general. Here, we comprehensively review the mechanisms that regulate the macrophage phagocytosis of GBM cells.

Mast cell density in Merkel cell carcinoma and its correlation with prognostic features and MCPyV status: a pilot study

Merkel cell carcinoma (MCC) is a rare, highly aggressive, primitive neuroendocrine carcinoma of the skin, the origin of which is not yet fully understood. Numerous independent prognostic factors have been investigated in an attempt to understand which are the most important parameters to indicate in the histological diagnostic report of MCC. Of these, mast cells have only been studied in one paper before this one. We present a retrospective descriptive study of 13 cases of MCC, received at the Department of Pathology over a 20-year period (2003–2023 inclusive) on which we performed a study using whole-slide (WSI) morphometric analysis scanning platform Aperio Scanscope CS for the detection and spatial distribution of mast cells, using monoclonal anti-tryptase antibody and anti-CD34 monoclonal antibody to study the density of microvessels. In addition, we analyzed MCPyV status with the antibody for MCPyV large T-antigen (Clone CM2B4). We found statistically significant correlation between mast cell density and local recurrence/distant metastasis/death-of-disease (p = 0.008). To our knowledge, we firstly reported that MCPyV ( −) MCC shows higher mast cells density compared to MCPyV ( +) MCC, the latter well known to be less aggressive. Besides, the median vascular density did not show no significant correlation with recurrence/metastasis/death-of-disease, (p = 0.18). Despite the small sample size, this paper prompts future studies investigating the role of mast cell density in MCC.

Enhanced treatment of breast cancer brain metastases with oncolytic virus expressing anti-CD47 antibody and temozolomide

Limited therapeutic options are available for patients with breast cancer brain metastases (BCBM), and thus there is an urgent need for novel treatment approaches. We previously engineered an effective oncolytic herpes simplex virus-1 (oHSV) expressing a full-length anti-CD47 monoclonal antibody (mAb) with a human IgG1 scaffold (OV-αCD47-G1) that was used to treat both ovarian cancer and glioblastoma. Here, we demonstrate that the combination of OV-αCD47-G1 and temozolomide (TMZ) improve outcomes in preclinical models of BCBM. The combination of TMZ with OV-αCD47-G1 synergistically increased macrophage phagocytosis against breast tumor cells and led to greater activation of NK cell cytotoxicity. Additionally, the combination of OV-αCD47-G1 with TMZ significantly prolonged the survival of tumor-bearing mice when compared to TMZ or OV-αCD47-G1 alone. Combination treatment with the mouse counterpart of OV-αCD47-G1, termed OV-A4-IgG2b, also enhanced mouse macrophage phagocytosis, NK cell cytotoxicity, and survival in an immunocompetent model of mice bearing BCBM compared to TMZ or OV-A4-IgG2b alone. Collectively, these results suggest that OV-αCD47-G1 combined with TMZ should be explored in patients with BCBM.

Updated safety, efficacy, pharmacokinetics, and biomarkers from the phase 1 study of IMC-002, a novel anti-CD47 monoclonal antibody, in patients with advanced solid tumors.

Updated safety, efficacy, pharmacokinetics, and biomarkers from the phase 1 study of IMC-002, a novel anti-CD47 monoclonal antibody, in patients with advanced solid tumors.

Mitochondria targeted drug delivery system overcoming drug resistance in intrahepatic cholangiocarcinoma by reprogramming lipid metabolism

The challenge of drug resistance in intrahepatic cholangiocarcinoma (ICC) is intricately linked with lipid metabolism reprogramming. The hepatic lipase (HL) and the membrane receptor CD36 are overexpressed in BGJ398-resistant ICC cells, while they are essential for lipid uptake, further enhancing lipid utilization in ICC. Herein, a metal-organic framework-based drug delivery system (OB@D-pMOF/CaP-AC, DDS), has been developed. The specifically designed DDS exhibits a successive targeting property, enabling it to precisely target ICC cells and their mitochondria. By specifically targeting the mitochondria, DDS produces reactive oxygen species (ROS) through its sonodynamic therapy effect, achieving a more potent reduction in ATP levels compared to non-targeted approaches, through the impairment of mitochondrial function. Additionally, the DDS strategically minimizes lipid uptake through the incorporation of the anti-HL drug, Orlistat, and anti-CD36 monoclonal antibody, reducing lipid-derived energy production. This dual-action strategy on both mitochondria and lipids can hinder energy utilization to restore drug sensitivity to BGJ398 in ICC. Moreover, an orthotopic mice model of drug-resistant ICC was developed, which serves as an exacting platform for evaluating the multifunction of designed DDS. Upon in vivo experiments with this model, the DDS demonstrated exceptional capabilities in suppressing tumor growth, reprogramming lipid metabolism and improving immune response, thereby overcoming drug resistance. These findings underscore the mitochondria-targeted DDS as a promising and innovative solution in ICC drug resistance.

Abstract 6433: Anti-CD36 monoclonal antibody can sensitize the neoadjuvant chemotherapy response in PDAC by eliminating the senescent tertiary lymphatic structure

Abstract Objective: Pancreatic ductal adenocarcinoma (PDAC) is deadly and aggressive. Neoadjuvant chemotherapy (NAC) may reduce tumor volume and alter the resectability of the tumor. The efficacy of neoadjuvant chemotherapy in patients is still limited. Tertiary lymphoid structures (TLSs) develop in peripheral non-lymphoid tissues and are essential for the immune response and correlate with chemotherapy in various tumor types. However, more research is needed to determine how TLSs affect neoadjuvant chemotherapy in PDAC. We aim to determine how neoadjuvant chemotherapy affects tumor-infiltrating lymphocytes (TLSs) in PDAC patients. We also want to find a biomarker to boost PDAC neoadjuvant chemotherapy response. Methods: We retrospectively collected formalin-fixed paraffin embedded (FFPE) tissues from 115 PDAC patients without preoperative treatment and 72 patients pretreated with neoadjuvant chemotherapy. Multispectral images were processed using AI-powered image analysis software (Tissue Gnostics; Zeiss, Germany). The 10X Genomics platform was used to sequence single cells in tissue samples from five patients who received neoadjuvant chemotherapy (NAC) and five patients who had not received any treatment before analysis (pretreatment naïve, PN). TLS was separated using Laser Microdissection. The mouse model was created by orthotopically injecting tumor cells into the pancreas. Results: Patients who received NAC have a senescent microenvironment. High-throughput single-cell sequencing showed that the senescent gene signature score was enriched in the NAC group, especially in immune cells. The percentage of p16+ CD20+ B cells and P16+ CD3+ T cells in TLSs also increased in the NAC group. Spatial transcriptomics analysis exhibited that CD36 is highly expressed in senescent TLSs, which suggested that CD36 may be a marker for senescent TLSs. Moreover, CD36+ TLSs induced the infiltration of MDSCs and regulatory T cells. CD36+ TLSs were an independent prognostic factor for poor overall survival (OS) (HR=0.67, p=0.002) and progression-free survival (PFS) (HR=0.71, p=0.003). A mice orthotopic xenograft model showed that an anti-CD36 monoclonal antibody eliminated senescent TLSs in pancreatic tissue. MDSCs and Tregs in the surrounding tissue also decreased. Additionally, in vivo KPC mice model showed that the combination of an anti-CD36 and chemotherapy reduced tumor volume and increased survival compared to chemotherapy alone. Conclusion: NAC has the potential to induce the senescence of tumor-associated TLSs, a phenomenon that has been linked to unfavorable prognosis in PDAC. CD36 serves as a viable marker for senescent TLSs that can be targeted. The administration of an anti-CD36 monoclonal antibody can potentially enhance the efficacy of chemotherapy in PDAC by selectively eliminating CD36+ senescent TLSs in mouse models. Citation Format: Jingrui Yan, Tianxing Zhou, Chao Yang, Yongjie Xie, Jun Yu, Jihui Hao. Anti-CD36 monoclonal antibody can sensitize the neoadjuvant chemotherapy response in PDAC by eliminating the senescent tertiary lymphatic structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6433.

Abstract 5244: SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models

Abstract The CD47-SIRPα axis is a critical checkpoint that prevents SIRPα-positive macrophages from phagocytosing CD47-expressing solid tumors. Several agents aiming to block this axis have recently entered early clinical trials including anti-CD47 and anti-SIRPα monoclonal antibodies (mAb). These checkpoint inhibitors (CPI) aim to modulate the phagocytotic activity of endogenous tumor associated macrophages (TAMs). However, the adoptive transfer of macrophages resistant to CD47-based inhibition in the tumor microenvironment (TME) could also increase clinical efficacy while avoiding side effects linked to the use of anti-CD47 mAb. Cell therapy based on autologous macrophages have gained increasing attention for cancer treatment due to their ability to infiltrate into the immunosuppressive TME and their unique immunomodulatory characteristics. However, due to required intricate genetic manipulation of autologous macrophage cell product for each patient, optimization and consistency of the cell product remains challenging. In contrast, the use of induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) facilitates the introduction of genetic modifications to further optimize the iMAC cell product and limits the need for combination therapies. The knockout (KO) of the SIRPα gene in iMACs is a promising genetic modification that results in a potent iMAC cell therapy product resistant to phagocytosis inhibition by CD47-expressing tumor cells. A SIRPα KO was introduced in a fully characterized GMP iPSC line that was then differentiated to iMACs using Evotec’s 3D differentiation protocol. The SIRPα KO iMACs were then evaluated for their antibody-dependent cellular phagocytosis (ADCP) capacity in comparison to antibody-loaded wildtype (WT) iMACs when co-cultured with CD47-expressing tumor cells. SIRPα KO iPSCs showed differentiation potential comparable to WT iPSC and resulted in iMACs expressing typical markers of fully differentiated macrophages. SIRPα KO iMACs exhibited increased phagocytic potency and killing capacity compared to WT iMACs when both cell types were exposed to CD47-positive tumor cells and loaded with the same tumor-targeting mAb. This increased phagocytosis of tumor cells by antibody-loaded SIRPα KO iMACs was also comparable to ADCP observed for WT iMACs in the presence of an anti-CD47 blocking antibody. Using Evotec’s gene editing platform we were able to efficiently generate SIRPα-deficient iPSCs that serve as the starting material to manufacture highly pure, genetically modified iMACs that lack SIRPα expression rendering them resistant to CD47-dependent inhibition of phagocytosis. This novel allogeneic off-the-shelf iMAC cell product overcomes the need to combine this cell therapeutic with CD47-SIRPα axis CPIs and provides the basis to develop innovative treatments for solid tumors. Citation Format: Kathrin Haake, Michela Mirenda, Quentin Bernard, Philip Hublitz, Lucie Gouxette, Martin Briscadieu, Philine Scheinpflug, Garima Singh, Alica Hinkelmann, Tanja Schneider, Michael Esquerré, Audrey Holtzinger, Michael Epstein, Daniel Sommermeyer, Michael Paillasse, Andreas Scheel, Markus Dangl, Monika Braun, Nadja Wagner. SIRPα knockout iPSC-derived macrophages (iMACs) are resistant to CD47-dependent inhibition of phagocytosis and efficiently kill tumor cells in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5244.

Abstract 1857: Development of monoclonal antibodies against CD44v specifically expressed in cancers

Abstract CD44 is a transmembrane glycoprotein that binds to the extracellular matrix including hyaluronic acid. Human CD44 has 19 exons, 10 of which are commonly present in all isoforms. CD44 standard isoform (CD44s) is composed of the common exons, and is expressed in many cell types. CD44 variants (CD44v) are produced by alternative splicing, in which the remaining nine variant exons are inserted into the CD44 standard isoform in various combinations. The CD44 isoforms have overlapping and unique functions. Both CD44s and CD44v harbor hyaluronan-binding motifs that promote interaction with the tumor microenvironment, which activate various signaling pathways in tumor cells. CD44v is mainly expressed in epithelial cells and is involved in cell adhesion, proliferation, and survival. Overexpression of CD44v is observed in many solid tumors and plays an important role in invasion, metastasis, and acquisition of stemness. In this study, we employed the Cell-Based Immunization and Screening method (CBIS method), and established highly sensitive anti-CD44 monoclonal antibodies (mAbs) using CD44v3-10-overexpressed cell lines as immunogens. After that, we determined the epitopes by enzyme-linked immunosorbent assay and investigated their applications. As a result, we successfully established eight mAbs against each CD44v (v3 to v10), which can be used for flow cytometry, western blotting, and immunohistochemistry. Furthermore, some of them exhibited cancer specificity against pancreatic cancers, colorectal cancers, gastric cancers, or oral cancers, indicating that established anti-CD44v mAbs are useful for the antibody therapies. Citation Format: Yukinari Kato, Hiroyuki Suzuki, Mika K. Kaneko. Development of monoclonal antibodies against CD44v specifically expressed in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1857.

Gentulizumab, a novel anti-CD47 antibody with potent antitumor activity and demonstrates a favorable safety profile

BackgroundTargeting CD47/SIRPα axis has emerged as a promising strategy in cancer immunotherapy. Despite the encouraging clinical efficacy observed in hematologic malignancies through CD47-SIRPα blockade, there are safety concerns related to the binding of anti-CD47 antibodies to CD47 on the membrane of peripheral blood cells.MethodsIn order to enhance the selectivity and therapeutic efficacy of the antibody, we developed a humanized anti-CD47 monoclonal antibody called Gentulizumab (GenSci059). The binding capacity of GenSci059 to CD47 was evaluated using flow cytometry and surface plasmon resonance (SPR) methods, the inhibitory effect of GenSci059 on the CD47-SIRPα interaction was evaluated through competitive ELISA assays. The anti-tumor activity of GenSci059 was assessed using in vitro macrophage models and in vivo patient-derived xenograft (PDX) models. To evaluate the safety profile of GenSci059, binding assays were conducted using blood cells. Additionally, we investigated the underlying mechanisms contributing to the weaker binding of GenSci059 to erythrocytes. Finally, toxicity studies were performed in non-human primates to assess the potential risks associated with GenSci059.ResultsGenSci059 displayed strong binding to CD47 in both human and monkey, and effectively inhibited the CD47-SIRPα interaction. With doses ranging from 5 to 20 mg/kg, GenSci059 demonstrated potent inhibition of the growth of subcutaneous tumor with the inhibition rates ranged from 30.3% to complete regression. Combination of GenSci059 with 2.5 mg/kg Rituximab at a dose of 2.5 mg/kg showed enhanced tumor inhibition compared to monotherapy, exhibiting synergistic effects. GenSci059 exhibited minimal binding to hRBCs compared to Hu5F9-G4. The binding of GenSci059 to CD47 depended on the cyclization of N-terminal pyroglutamic acid and the spatial conformation of CD47, but was not affected by its glycosylation modifications. A maximum tolerated dose (MTD) of 450 mg/kg was observed for GenSci059, and no significant adverse effects were observed in repeated dosages up to 10 + 300 mg/kg, indicating a favorable safety profile.ConclusionGenSci059 selectively binds to CD47, effectively blocks the CD47/SIRPα axis signaling pathway and enhances the phagocytosis effects of macrophages toward tumor cells. This monoclonal antibody demonstrates potent antitumor activity and exhibits a favorable safety profile, positioning it as a promising and effective therapeutic option for cancer.

HDAC Inhibition Induces CD26 Expression on Multiple Myeloma Cells via the c-Myc/Sp1-mediated Promoter Activation.

There is a desire to induce and sustain CD26 expression on multiple myeloma cells to elicit superior anti-myeloma response by humanized anti-CD26 mAb therapy. HDACi upregulates the expression levels of CD26 on myeloma cells via the increased acetylation of c-MycK323 on the CD26 promoter, leading to initiation of CD26 transcription, thereby synergistically augments the efficacy of CD26mAb against CD26neg myeloma cells.

Pretreatment with Daudi cells eliminates anti-CD47 monoclonal antibody interference in immunohematology testing.

Anti-CD47 monoclonal antibodies have increasing clinical applications in the treatment of cancer. However, anti-CD47 monoclonal antibodies interfere with immunohematology testing in patients who require blood transfusion. As the current approaches to removing any interferences have technical problems, new methods need to be developed to resolve anti-CD47 interference in immunohematology testing. We evaluated the Daudi cell line for the adsorption of free anti-CD47 monoclonal antibodies from patients' plasma to facilitate immunohematology testing in patients treated with anti-CD47 monoclonal antibody. CD47 expression was identified on the Daudi cells using flow cytometry and confocal microscopy. Next, we tested the ability of intact Daudi cells mixed with simulating plasma and clinical samples to achieve efficient removal of interfering anti-CD47 monoclonal antibodies. The indirect antiglobulin test was used to verify whether interference from anti-CD47 monoclonal antibodies in plasma was eliminated and whether the detection of other irregular antibodies was affected. The effect of eliminating interference was also investigated in relation to the time that the Daudi cells were stored after having been fixed with paraformaldehyde. CD47 expression was higher on Daudi cells than on red blood cells. Analysis of the indirect antiglobulin test results revealed that anti-CD47 monoclonal antibody-treated patients' plasma absorbed by Daudi cells for 15 min at 37°C could completely prevent the interference of anti-CD47 monoclonal antibodies in immunohematology testing while the detection of the tested antibodies, including anti-D and anti-K, was unaffected. By decreasing the incubation time, we discovered that interferences in samples with agglutination strengths below 2+ could be eliminated after incubation for 5 min. Of importance, Daudi cells can be preserved with 4% paraformaldehyde for 14 days as short-term storage reagents. This is the first study in which Daudi cells were used to effectively resolve the interference of anti-CD47 monoclonal antibodies in pretransfusion tests.

The Potent and Selective Antitumor Activity of CO-1 and Fully Humanized CO-1bi in Treatment of B Cell Non-Hodgkin's Lymphoma

Recent advancements have identified CD47 blockade as a promising approach for the treatment of B cell Non-Hodgkin's Lymphoma (B-NHL). CD47 blockade inhibits the CD47-SIRPα axis, which inhibits a “don't eat me” signal that facilitates the phagocytosis of tumor cells by macrophages [1,2]. While the effect of anti-CD47 monoclonal antibodies (mAbs) as monotherapy has been limited in B-NHL, combining CD47 mAbs with conventional treatment such as rituximab has demonstrated more encouraging outcomes. This combinatory approach may be attributed to the need for additional cytotoxic mechanisms to achieve clinically meaningful responses with CD47-mediated therapy in B-NHL [3]. Additionally, CD47 blockage has raised concerns about off-target effects and red blood cell depletion [4]. To address these challenges and improve treatment possibilities we developed a fully humanized CO-1bi based on the chimeric CO-1. The bifunctional CO-1 is an agonistic IgG4 anti-CD47 mAb designed to induce direct and rapid programmed cancer cell death (PCCD) and stimulate phagocytosis through blockage of CD47-SIRPα binding. CO-1bi is a humanized bivalent scFv-Fc fusion of CO-1 with superior safety (no red blood cell hemagglutination) while maintaining the functionality of CO-1. In this study, we evaluated the antitumor activity of CO-1 and CO-1bi on different B-NHL cell lines in vitro as well as in xenografts of B-NHL established in NOD- scid IL2Rγ null (NSG) mice. Both constructs demonstrated potent and fast cell death induction in lymphoma cell lines while sparing normal B cells. The robust antitumor activity demonstrated by CO-1 and CO-1bi as single agents in xenograft models of Burkitt's lymphoma underscores the significance of their additional agonistic PCCD capabilities compared to other anti-CD47 agents. The emergence of CO-1bi offers a unique strategy with dual functionality for the treatment of B-NHL and other malignancies without red blood cell depletion characterized by current anti-CD47 mAb in clinical trials. This innovative approach provides an improved and promising avenue for cancer treatment.

SAMHD1 Inhibitor Combined with Immune Checkpoint Inhibitor Elicits an Anti-AML Immunity

Despite advances in targeted therapy and use of molecular/cytogenetic risk stratification to guide treatment, clinical outcomes for patients with acute myeloid leukemia (AML) remain unfavorable. Harnessing the immune system with CAR-T or immune checkpoint inhibitors (ICIs) has been proven successful in other hematological malignancies and solid tumors. The use of these immunotherapies for the AML treatment remains challenging due to lack of selective target antigens or low expression of immune checkpoint proteins. Recently, one strategy proposed to transform AML from immune-cold to immune-hot is to trigger a type-I interferon (IFN-I) response via a dsDNA related cGAS/STING signaling or dsDNA related RIG-I/MDA5 signaling. We recently reported that the dNTP and Ara-CTP (active form of cytarabine [AraC]) hydrolase SAMHD1, whose expression levels are elevated in AML patients relative to normal donors, limits IFN-I and T cell responses in murine AML model ( Blood, 2022, 140 [Supplement 1]: 679-680). We also found that innate immune activation seen following SAMHD1 knockdown is associated with its catalytic activity inhibition in cancer cells, providing a rationale for the development of SAMHD1 inhibitor. To identify SAMHD1 inhibitor, we conducted a virtual screen on 8,000,000 compounds based on SAMHD1 crystal structure (PBD 6TXC). The top 500 hits were then assessed in SAMHD1-WT- or H233A (SAMHD1 loss-of-function mutant) -expressing Molm13 cells using a phenotypic screen for AraC sensitization. Specifically, a SAMHD1 inhibitor should sensitize SAMHD1-WT (S-WT) but not H233A (S-H233A) cells to AraC treatment. Among the top 20 hits from phenotypic screen, our HPLC-based cell-free and cell-based SAMHD1 enzymatic assay revealed Samin27 as the most potent hit in blocking SAMHD1 hydrolysis and promoting AraCTP accumulation. Therefore, we assessed the efficacy of Samin27 combined with AraC in vivo. Samin27 administration (25mg/kg i.p. qd, starting on day 8 for 7 days) enhanced AraC efficacy (50mg/kg i.p. qd, starting 8th day for 5 days) in NSG mice implanted with Molm13 cells, as evidenced by extended survival relative to AraC alone. Moreover, the physical interaction between Samin27 and SAMHD1 protein were confirmed in a Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance (NMR) titration assay. RNA-seq results revealed Samin27 ex-vivo treatment remarkably upregulated IFN-I response genes in Molm13 cells, consistent with transcriptome results of SAMHD1-knockdown (S-KD) Molm13 cells. SAMHD1 activity is known to restrict replication of LINE-1 (L1), which is the only autonomously active transposable elements (TE) in mammals; elevated retrotransposition induces DNA damage and promotes cytoplasmic DNA accumulation, resulting in an IFN-I response via cGAS signaling. Next, to explore the mechanism underlying SAMHD1-inhibition induced immunity, we asked if SAMHD1 loss promoted retrotransposition and upregulated TE transcripts in leukemia cells. Through re-analysis of RNA-seq data in Molm13 cells, we found SAMHD1 inhibition upregulated most L1 elements and LTR-containing endogenous retrovirus (ERV) subfamilies. To determine if Samin27 induced immunity via L1 upregulation, we employed 3TC treatment (3TC, the nucleoside reverse transcriptase inhibitor) to block L1 upregulation. 3TC treatment blocked Samin27-mediated IFN-I response genes upregulation. Given that IFN-I response can stimulate innate immune cells (macrophage and T cell) cross-priming, potentially rendering AML cells more susceptible to anti-CD47 monoclonal antibody (aCD47) treatment, we then assessed whether combining a SAMHD1 inhibitor with an aCD47 act synergistically. We first implanted murine MLL-AF9 (MA9) leukemia cells into WT B6 mice. Then we treated leukemic mice with isotype control (Ctrl), anti-CD47 (BE0283 [BioXCell], 10mg/kg, i.p. qd, 3 weeks), Samin27 (25mg/kg i.p. qd, 3 weeks) or combination. Notably, pharmacological inhibition of SAMHD1 remarkably inhibited leukemia progression, decreasing leukemia burden (Fig. A.B). In another cohort, combination treatment extended mouse survival (Fig. C). Collectively, our results may provide a rationale for combining Samin27 with aCD47 against AML population, where single anti-CD47 treatment shows little effects.

Transfusion management in the era of magrolimab (Hu5F9-G4), an anti-CD47 monoclonal antibody therapy.

Transfusion management in the era of magrolimab (Hu5F9-G4), an anti-CD47 monoclonal antibody therapy.

TMIC-24. FERROPTOSIS INDUCES IMMUNOGENIC CELL DEATH AND SYNERGIZES WITH CD47 BLOCKADE TO REPROGRAM THE GLIOMA MICROENVIRONMENT

Abstract Immunotherapy has thus far proven ineffective in glioblastoma, in part due to the immune cold tumor microenvironment. Here we demonstrate that ferroptosis specifically targets a quiescent, immunosuppressive astrocyte-like glioma population and can therefore be used as a novel inducer of immunogenic cell death to repolarize the immune microenvironment by promoting tumor cell phagocytosis and antigen presentation. First, we characterized the immunogenic effects of ferroptosis by using a glioma cell-myeloid cell co-culture system and patient-derived organotypic slice cultures and performed phagocytosis assays, flow-cytometry, immunohistochemistry, cytokine arrays and single cell RNAseq. GPX4-dependent ferroptosis induced translocation of the pro-phagocytic eat-me signal calreticulin in both murine glioma cells and human tissue samples. Ferroptotic-driven tumor cell death reprogramed local myeloid populations to promote glioma phagocytosis, MHCII-driven antigen presentation, and paracrine cytokine signaling involved in T-cell recruitment. Local delivery of RSL3, a GPX4 inhibitor, in a murine glioma model polarized myeloid cells to a pro-phagocytic phenotype and significantly increased intra-tumoral levels of CD4+ and CD8+ T-cells. Astrocytic-like tumor cells upregulate CD47 as a mechanism to evade phagocytosis. Therefore, we combined ferroptosis with an anti-CD47 monoclonal antibody (aCD47) to enhance reprogramming of the immune cold microenvironment. In an orthotopic murine glioma model, adding aCD47 to RSL3 led to significantly increased tumor phagocytosis, antigen presentation and prolonged survival. Thus, we demonstrate that local delivery of ferroptosis-inducing agents in conjunction with CD47 blockade is an effective multi-modality therapeutic regimen capable of reversing the immunosuppressive glioma microenvironment.